Comprehensive protease specificity profiling

Protease enzymes are of great importance in medicine, industry, and as research tools. Despite the crucial need for detailed knowledge of their proteolytic cleavage specificity, many proteases are poorly characterized. We present a method for fully characterizing the cleavage specificity of proteases through the comprehensive profiling of all possible permutations of octamer peptide substrates in a single experiment. The powerful combination of in vitro selection with high-throughput sequencing, mass spectrometry, and automated motif mining enabled the screening of mixtures of >10 ¹² peptides. We developed freely available software that easily integrates the massive amounts of cleavage data into user-friendly specificity information. We applied this method to three different proteases that had either narrow (factor Xa) or broad specificity (ADAM17 and streptopain). The resulting specificity maps revealed motifs that corroborate canonical known cleavage sites, yet step further into extended spectrum preferences and yield insights into the function of broad specificity proteases.